Perylene‐Mediated Electron Leakage in Respiratory Chain to Trigger Endogenous ROS Burst for Hypoxic Cancer Chemo‐Immunotherapy

Abstract Perylene derivatives can be stimulated by the hypoxic tumor microenvironment to generate radical anion that is proposed to arouse electron exchange with oxidizing substance, and in turn, realize reactive oxygen species (ROS) burst. Here, three perylene therapeutic agents, PDI‐NI, PDIB‐NI, and PDIC‐NI, are developed and it is found that the minimum lowest unoccupied molecular orbital (LUMO) energy level makes PDIC‐NI most easily accept electrons from the oxidative respiratory chain to form lots of anions, and the resultant maximum ROS generation, establishing an unambiguous mechanism for the formation of perylene radical anions in the cell, presents solid evidence for LUMO energy level determining endogenous ROS burst. Stirringly, PDIC‐NI‐induced ROS generation arouses enhanced mitochondrial oxidative stress and concurrently activates immunogenic cell death (ICD), which not only efficiently kills lung tumor cells but also reprograms immunosuppressive tumor microenvironment, including the cytokine secretion, dendritic cell maturation, as well as cytotoxic T lymphocytes activation, to inhibit the growth of xenografted and metastasis tumor, presenting a proof‐of‐concept demonstration of perylene that acts as an integrated therapeutic agent to well realize hypoxia‐activated chemotherapy with ICD‐induced immunotherapy on lung cancer.

Protein extraction. PDIC-NI solution (1 or 2 μg mL -1 ) was added to the cell culture medium, which incubated with the cells for 8 h. After washed with PBS for 3 times, The cells were collected following by addition of cell lysate (500 μL), then the cells were quickly scraped off with a cell scraper and transferred to a new EP tube. Later, the tube was kept in ice for 30 min to make the cells fully lysed. After centrifuged at 12,000 g for 15 min (4 °C), the supernatant was put into another new EP tube and determined to quantitative concentration through BCA protein quantification method.
Western blotting assay. Different electrophoretic concentrates were prepared according to the molecular weight of proteins. The extracted protein in each group was injected into each hole (20 μL) for gel electrophoresis. After full electrophoresis, the protein was transferred to PVDF membrane. 5% free-fat milk was applied to block nonspecific antigen of PVDF membrane in TBST solution for 3 h. The washed membrane was cut into strips according to the molecular weight of the target protein, and then incubated with the corresponding primary antibody overnight (4 °C). The primary antibody was removed in the next day, and the membrane was incubated with the secondary antibody at room temperature for 2 h. Protein bands can be displayed by ECL imaging method.
Analysis of mitochondrial complex expression. The extracted protein in each group was collected and levels of mitochondrial complex were tested by ELISA kits according to standard schemes. Simultaneously, the expression of mitochondrial complexes in tumor cells was further analyzed by western blotting assay.
Intracellular Ca 2+ level detection. Cells in logarithmic growth phase were treated with PDIC-NI (1 or 2 μg mL -1 ) for 8 h. Cells were harvested, centrifuged and then rinsed with PBS to prepare single cell suspensions, and stained with Fluo-4 AM. Cells were cultured at 37 °C (30 min) and examined by CytoFLEX flow cytometer and analyzed with Flow J software.
Cyclophilin D protein detection. After cells were completely adherent to the wall in 20 mm culture dish, PDIC-NI solution (1 or 2 μg mL -1 ) was putted in and continued to culture for 8 h.
Then, Mitotracker green was injected into DMEM medium (10 nM) to prepare working solution, subsequently added into the culture dish to incubate with cells at 37 °C (30 min).
Next, different groups of cells were washed by precooling PBS and fixed with 4% paraformaldehyde at 37 ℃ (30 min). Subsequently, cells were incubated with PBS containing 0.2% Triton X-100 (10 min). Immediately, 5% free-fat milk was used to block with the cells at room temperature (2 h). Primary antibody was incubated with the cells at 4 °C overnight and then was removed in the next day. Finally, the secondary antibody (1:500 dilution) of goat anti-rabbit IgG-Alexa 594 was added in each group for 2 h at room temperature.
Confocal laser microscopy was used to observe and take pictures. And the co-localization of these were calculated by image J software.
Apoptosis detection experiment. Apoptosis was detected by flow cytometry. PDIC-NI (1 or 2 μg mL -1 ) was applied to Cells in logarithmic growth phase (24 h 7 The release of ATP detection. After cells were completely adherent to the wall in 20 mm culture dish, PDIC-NI solution (1 or 2 μg mL -1 ) was putted in and continued to culture for 12 h. Then the culture medium was collected to the following test. After 100 μL ATP detection working solution and 20 μL medium was added into each well, and the fluorescence intensity was detected by Microplate Spectrophotometer. The standard curve of ATP was constructed with fluorescence intensity as longitudinal axis and ATP concentration as lateral axis. The fluorescence values of each sample were taken into the standard curve to calculate ATP concentration.
In vivo subcutaneous tumor models. All animal experiments have been approved by the Animal Management and Ethics Committee of Henan University (Nos: HUSOM-2021-001).
The 5-week-old C57BL/6 mice were raised in SPF animal room. A suspension of 5×10 5 LLC cells in PBS solution was inoculated subcutaneously in the right lower limb of mice. LLC tumor-bearing model were randomized into control group and treatment group (4 mice in each group). After the tumor grew to 100 mm 3 , the control group was treated with physiological saline, and the treatment group was treated with 2 mg kg -1 PDIC-NI every 2 days for 5 times.
The change of tumor volume and weight of mice were recorded every 2 days. After the treatment, the mice were euthanized, and the eyeball blood, main organs and tumor tissues were collected for analyses, and then the eyeball blood was collected to test the cytokines and other indicators while the tissues were fixed for subsequent experiments.
In vivo lung metastasis models. The 5-week-old C57BL/6 mice were injected intravenously with 4×10 5 LLC cancer cells. After 14 days, mice were randomized into the PBS group, oxaliplatin group and PDIC-NI group (4 mice in each group). Mice were injected with PBS, oxaliplatin or PDIC-NI solution (2 mg kg -1 ) through caudal vein every 3 days for 18 days. The weight of mice was recorded every 3 days.
Cytokines detection. After the treatment, mice blood was collected after anesthesia and placed in tubes containing anticoagulant heparin sodium and serum was centrifuged for analysis.
8 ELISA kits were used to detect the concentration of IFN-ɣ, and TNF-α based on the standard protocols.
Flow cytometric analysis the Ex vivo immune response. In order to assess the level of DC Subsequently, the tissues were sectioned by paraffin section machine, stained with hematoxylin and eosin and photographed by microscope.
Immunohistochemical staining (Ki67). Tumor tissues was taken and fixed in 4% formaldehyde solution (24 h). Then tissues were dehydrated in different concentrations of alcohol solution and embedded into paraffin. After blocking with 3% hydrogen peroxide (30 min), antigen retrieval was performed with citrate buffer (15 min) in microwave oven. Ki67 (1: 200) was added with slices overnight. Next, the sections were incubated with the secondary antibody (40 min) and stained by DAB Immunohistochemistry Color Development Kit. The whole section image was collected using fluorescence microscopy.
TUNEL staining. Tumor tissues were taken and fixed in 4% formaldehyde solution (24 h).
Then tissues were dehydrated in different concentrations of alcohol solution and embedded into paraffin. Next, the section was incubated with TUNEL staining in dark for 60 min, and observed and photographed by fluorescence microscopy.
Blood routine test. The blood was collected and then put in a 1.5 mL anticoagulant EP tube containing sodium heparin. An animal-specific blood gas and electrolyte analyzer analyzed the following indicators such as hemoglobin (HGB), lymphocytes (LYM), white blood cells (WBC), red blood cells (RBC), red blood cell distribution width (RDW), and so on.
Blood biochemical test. The blood was collected and placed in a biochemical incubator (30 min), and then centrifuged at 6000 rpm (10 min). The supernatant was used to detect the following indicators including alanine aminotransferase (ALT), aspartate aminotransferase (AST), serum creatine kinase (CK) and blood urea nitrogen (BUN).

Statistical analysis. Statistical differences between treatments were measured via GraphPad
Prism software. Student's t-test was applied for pairwise comparisons. p value less than 0.05 was regarded significant.  Figure S1. The 1 H NMR of PDI-NI. Figure S2. The 13 C NMR of PDI-NI. Figure S3. High resolution ESI-MS of PDI-NI. Figure S4. The 1 H NMR of PDIB-NI. Figure S5. The 13 C NMR of PDIB-NI.                  20 Figure S27. H&E staining images of main organs from LLC xenografted tumor-bearing mice.  23 Figure S32. Representative flow cytometry analysis and quantifications of regulatory T cells (CD4 + CD25 + Foxp3 + ) in lung metastasis tumor. Data represent mean ± SD (n = 3), t test versus control: